Throughout this application, various references are referred to in the text within parentheses in full or within parentheses by number. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for those references referred to by number may be found at the end of this application, preceding the claims.
The following standard abbreviations are used throughout to refer to amino acids:
TRAF-3 gene products are signaling molecules that interact with the cytoplasmic tails of CD40 (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995), other Tumor Necrosis Factor-Receptor (TNF-R) family members (e.g. LTxcex2-R, CD30, CD27, Ox40) (Mosialos et al., 1995; Gedrich et al., 1996; Boucher et al., 1997; Yamamoto et al., 1998; Vanarsdale et al., 1997; Arch and Thompson, 1998; Kawamata et al., 1998) and the Epstein-Barr virus latent membrane protein, LMP1 (Mosialos et al., 1995). The finding that TRAF-3(xe2x88x92/xe2x88x92) lymphocytes are specifically defective in T-B lymphocyte collaboration, indiates that TRAF-3 gene products are required for signaling events that underlie this function (Xu et al., 1996). Full-length TRAF-3 alone is unlikely to account for such signaling, since over-expression of full-length TRAF-3 fails to induce NF-xcexaB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, alternative splicing of TRAF-3 transcripts generates mRNA species that encode at least 3 putative isoforms with altered Zn finger domains that may participate in transmitting receptor signals to the nucleus (Sato et al., 1995; Krajewski et al., 1997; van Eyndhoven et al., 1998). Therefore, the present study addressed whether TRAF-3 mRNA splice-deletion variants encode TRAF-3 protein isoforms that are able to induce NF-xcexaB activation.
TRAF-3 is a member of the TRAF (TNF Receptor-associated factor) family of proteins, of which six have been identified (TRAF-1 through 6) (Rothe et al., 1994; Regnier et al., 1995; Ishida et al., 1996; Ishida et al., 1996; Kashiwada et al., 1998; Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). TRAF-3, like other TRAFs, appears to lack intrinsic catalytic activity, which suggests that TRAF-3 functions as a docking or adaptor molecule for other proteins that mediate signaling events. TRAF family members are related by significant homology in their carboxy-terminal TRAF-C domains (Rothe et al., 1994; Cheng et al., 1995). The TRAF-3 TRAF-C domain is known to be important for the interaction of TRAF-3 with the cytoplasmic tails of TNF-R family receptors (Cheng et al., 1995; Force et al., 1997; Vanarsdale et al., 1997), homo-oligomerization (Cheng et al., 1995; Force et al., 1997; Sato et al., 1995; Pullen et al., 1998) and binding to cytoplasmic proteins such as I-TRAF/TANK (Rothe et al., 1996; Cheng and Baltimore, 1996) and NIK (Song et al., 1997; Malinin et al., 1997). In addition to the TRAF-C domain, TRAF-3 contains an amino-terminal RING finger domain, five atypical Zn finger motifs, an iso-leucine zipper domain and a TRAF-N domain (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In TRAF-2 and TRAF-5, the RING finger and Zn finger domains have been shown to play important roles in mediating NF-xcexaB activation (Takeuchi et al., 1996; Dadgostar and Cheng, 1998). The functional potentials of the TRAF-3 RING finger and Zn finger domains remain enigmatic, since TRAF-3 itself fails to induce NF-xcexaB activation (Rothe et al., 1995; Takeuchi et al., 1996; Dadgostar and Cheng, 1998). However, the TRAF-3 RING finger domain is capable of supporting NF-xcexaB activation in chimeric TRAF-3/5 molecules (Dadgostar and Cheng, 1998).
Alteration of the TRAF-3 Zn finger domain by alternative mRNA splicing was suggested by analysis of the sequences of the initial TRAF-3 cDNA clones isolated. One TRAF-3 cDNA clone (CAP1) contains a 75 bp deletion, relative to 3 other TRAF-3 cDNA clones (termed CRAF1, CD40bp and LAP1) (Cheng et al., 1995; Hu et al., 1994; Sato et al., 1995; Mosialos et al., 1995). In addition, 2 other TRAF-3 mRNA variants containing 156 bp and 168 bp deletions were recently identified (van Eyndhoven et al., 1998). Each of the 3 mRNA species with deleted elements, encodes a putative TRAF-3 protein isoform with an altered number and composition of the Zn fingers. These putative isoforms have been termed, TRAF-3b (xcex9425aa), TRAF-3c (xcex9452aa) and TRAF-3d (xcex9456aa) (Krajewski et al., 1997; van Eyndhoven et al., 1998). Characterization of the human TRAF-3 genomic structure indicated that these 3 TRAF-3 mRNA species result from alternative mRNA splicing (van Eyndhoven et al., 1998). Further analysis of the TRAF-3 gene suggested that a large number of additional splice variants may be generated, since each of the splice junctions of exons 4 through 10 are class xe2x80x9c0xe2x80x9d. Therefore, splice-deletion variants involving any or all of exons 5-10 would maintain open reading frames (ORFs) (van Eyndhoven et al., 1998). However, additional splice-deletion variants have not been characterized and the functions of the identified or predicted splice-deletion variants have not been studied.
The present invention provides an isolated TRAF-3 deletion isoform encoded by the nucleic acid sequence (SEQ ID NO: 35) shown in FIG. 15 (deletion isoform xcex94130 nucleic acid). One embodiment of the present invention is an isolated TRAF-3 protein which comprises the sequence in FIG. 16 (SEQ ID NO: 36) (xcex94130 protein). Another embodiment of the present invention is an isolated TRAF-3 deletion isoform encoded by the nucleic acid sequence (SEQ ID NO: 37) shown in FIG. 17 (deletion isoform xcex94221 nucleic acid). A further embodiment of the present invention is an isolated TRAF-3 deletion isoform protein which comprises the sequence in FIG. 18 (SEQ ID NO: 38) (deletion isoform xcex94221 protein).
The present invention also provides for a method of inhibiting activation by CD40 ligand of cells expressing CD40 on the cell surface, comprising contacting the cells with a TRAF-3 deletion isoform comprising the sequence shown in FIG. 16 or 18, the isoform protein being present in an amount effective to inhibit activation of the cells.
The present invention also provides for a method for inhibiting an autoimmune response in a subject which comprises administering to the subject an effective amount of a TRAF-3 deletion isoform protein comprising the sequence shown in FIG. 16 or 18 in an effective amount to inhibit an autoimmune response in a subject.
The invention also provides a method for identifying an agent that inhibits CD40-mediated cellular signaling in a cell which comprises: (a) contacting the cell with an agent under conditions wherein CD40-mediated cell activation occurs; and (b) determining whether CD40-mediated signaling is inhibited in the cell in the presence of the agent so as to identify whether the agent inhibits CD40-mediated cellular signaling.